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Rhizen Pharmaceuticals is developing RP-6530, a PI3K delta and gamma dual inhibitor, for the potential oral treatment of cancer and inflammation In November 2013, a phase I trial in patients with hematologic malignancies was initiated in Italy ]\. In September 2015, a phase I/Ib study was initiated in the US, in patients with relapsed and refractory T-cell lymphoma. At that time, the study was expected to complete in December 2016

PI3K delta/gamma inhibitor RP6530 An orally active, highly selective, small molecule inhibitor of the delta and gamma isoforms of phosphoinositide-3 kinase (PI3K) with potential immunomodulating and antineoplastic activities. Upon administration, PI3K delta/gamma inhibitor RP6530 inhibits the PI3K delta and gamma isoforms and prevents the activation of the PI3K/AKT-mediated signaling pathway. This may lead to a reduction in cellular proliferation in PI3K delta/gamma-expressing tumor cells. In addition, this agent modulates inflammatory responses through various mechanisms, including the inhibition of both the release of reactive oxygen species (ROS) from neutrophils and tumor necrosis factor (TNF)-alpha activity. Unlike other isoforms of PI3K, the delta and gamma isoforms are overexpressed primarily in hematologic malignancies and in inflammatory and autoimmune diseases. By selectively targeting these isoforms, PI3K signaling in normal, non-neoplastic cells is minimally impacted or not affected at all, which minimizes the side effect profile for this agent. Check for active clinical trials using this agent. (NCI Thesaurus)

RP6530 is a potent and selective dual PI3Kδ/γ inhibitor that inhibited growth of B-cell lymphoma cell lines with a concomitant reduction in the downstream biomarker, pAKT. Additionally, the compound showed cytotoxicity in a panel of lymphoma primary cells. Findings provide a rationale for future clinical trials in B-cell malignancies.

Mechanism of Action

While alpha and beta isoforms are ubiquitous in their distribution, expression of delta and gamma is restricted to circulating hematogenous cells and endothelial cells. Unlike PI3K-alpha or beta, mice lacking expression of gamma or delta do not show any adverse phenotype indicating that targeting of these specific isoforms would not result in overt toxicity. Dual delta/gamma inhibition is strongly implicated as an intervention strategy in allergic and non-allergic inflammation of the airways and other autoimmune diseases. Scientific evidence for PI3K-delta and gamma involvement in various cellular processes underlying asthma and COPD stems from inhibitor studies and gene-targeting approaches. Also, resistance to conventional therapies such as corticosteroids in several COPD patients has been attributed to an up-regulation of the PI3K delta/gamma pathway. Disruption of PI3K-delta/gamma signalling therefore provides a novel strategy aimed at counteracting the immuno-inflammatory response. Due to the pivotal role played by PI3K-delta and gamma in mediating inflammatory cell functionality such as leukocyte migration and activation, and mast cell degranulation, blocking these isoforms may also be an effective strategy for the treatment of rheumatoid arthritis as well.

Given the established criticality of these isoforms in immune surveillance, inhibitors specifically targeting the delta and gamma isoforms would be expected to attenuate the progression of immune response encountered in airway inflammation and rheumatoid arthritis.

Clinical Trials

Rhizen has identified an orally active Lead Molecule, RP-6530, that has an excellent pre-clinical profile. RP-6530 is currently in non-GLP Tox studies and is expected to enter Clinical Development in H2 2013.

In December 2013, Rhizen announced the start of a Phase I clinical trial. The study entitled A Phase-I, Dose Escalation Study to Evaluate Safety and Efficacy of RP6530, a dual PI3K delta /gamma inhibitor, in patients with Relapsed or Refractory Hematologic Malignancies is designed primarily to establish the safety and tolerability of RP6530. Secondary objectives include clinical efficacy assessment and biomarker response to allow dose determination and potential patient stratification in subsequent expansion studies.

Partners by Region

Rhizen’s pipeline consists of internally discovered (with 100% IP ownership) novel small molecule programs aimed at high value markets of Oncology, Immuno-inflammtion and Metabolic Disorders. Rhizen has been successful in securing critical IP space in these areas and efforts are on for further expansion in to several indications. Rhizen seeks partnerships to unlock the potential of these valuable assets for further development from global pharmaceutical partners. At present global rights on all programs are available and Rhizen is flexible to consider suitable business models for licensing/collaboration.

In 2012, Rhizen announced a joint venture collaboration with TG Therapeutics for global development and commercialization of Rhizen’s Novel Selective PI3K Kinase Inhibitors. The selected lead RP5264 (hereafter, to be developed as TGR-1202) is an orally available, small molecule, PI3K specific inhibitor currently being positioned for the treatment of haematological malignancies.

PATENT

Intermediate 1: 3-(3-fluorophenyl)-2-(l-hydroxypropyl)-4H-chromen-4-one: To a solution of 2-(l-bromopropyl)-3-(3-fluorophenyl)-4H-chromen-4-one1 (8.80 g, 24.36 mmol ) in DMSO (85 ml), n-butanol (5 ml) was added and heated to 120° C for 3h. The reaction mixture was cooled to room temperature (RT), quenched with water and extracted with ethyl acetate. The organic layer was dried over sodium sulphate and concentrated under reduced pressure. The crude product was purified by column chromatography with ethyl acetate: petroleum ether to afford the title compound as a yellow solid (2.10 g, 29 %) which was used without further purification in next step.

Intermediate 2: 3-(3-fluorophenyl)-2-propionyl-4H-chromen-4-one: DMSO (1.90 ml, 26.82 mmol) was added to dichloromethane (70 ml) and cooled to -78°C. Oxalyl chloride (1.14 ml, 13.41 mmol) was then added. After 10 minutes, intermediate 1 (2.00 g, 6.70 mmol) in dichloromethane (20 ml) was added dropwise and stirred for 20 min. Triethylamine (7 ml) was added and stirred for lh. The reaction mixture was quenched with water and extracted with dichloromethane. The organic layer was dried over sodium sulphate and concentrated under reduced pressure. The crude product was purified by column chromatography with ethyl acetate: petroleum ether to afford the title compound as a yellow liquid (1.20 g, 60%) which was used as such in next step.

Abstract

Background: Myelofibrosis (MF) represents a life-threatening neoplasm that manifests particularly in the elderly population and is characterized by bone marrow fibrosis and extramedullary hematopoeisis. While ruxolitinib, a JAK1/2 inhibitor, has recently been approved by the USFDA for its disease modifying potential in MF patients, it is still not considered as a curative option. Targeting another kinase such as PI3K, downstream of JAK, could therefore be a more efficient way of treating myelofibrotic neoplasms. RP6530 is a novel, potent, and selective PI3K δ/γ inhibitor that demonstrated high potency against PI3Kδ (IC50 = 25 nM) and γ (IC50 = 33 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. The objective of this study was to evaluate the effect of a combination of ruxolitinib and RP6530 in the JAK2-V617F mutant Human Erythroleukemia (HEL) cell line.

Methods: Passive resistance was conferred by incubating HEL cells with increasing concentrations of ruxolitinib over an 8-10-week period. Endogenous JAK2, PI3Kδ, PI3Kδ, and pAKT were estimated by Western Blotting. RP6530, ruxolitinib, and the combination of RP6530 + Ruxolitinib were tested for their effect on viability and apoptosis. Cell viability was assessed by a MTT assay. Induction of apoptosis was analyzed by Annexin V/PI staining.

Results: Resistance to ruxolitinib was confirmed by a right-ward shift in EC50 of ruxolitinib in a HEL cell proliferation assay (0.82 μM Vs. 12.2 μM). Endogeous pAKT expression was 3.7-fold higher in HEL-RR compared to HEL-RS cells indicating activation of the AKT signaling pathway. While single-agent activity of RP6530 was modest (33-46% inhibition @ 10 μM) in both HEL-RS and HEL-RR cells, addition of 10 μM RP6530 to ruxolitinib was synergistic resulting in a near-complete inhibition of proliferation (>90% for HEL-RS and >70% for HEL-RR). While the order of addition did not affect the potency of RP6530, addition of 5 μM RP6530, 4 h prior to the addition of ruxolitinib resulted in a significant reduction in EC50 of ruxolitinib (5.8 μM) in HEL-RR cells. On lines with cell proliferation data, incubation of 10 μM RP6530 with ruxolitinib for 72 h increased the percent of apoptotic cells (55% in HEL-RS and 37% in HEL-RR) compared to either agent alone (16-27% in HEL-RS and 17-21% in HEL-RR).

Conclusions: Ruxolitinib resistance in the V617F JAK-2 mutant HEL cells is accompanied by an increase in pAKT expression. Inhibition of pAKT via the addition of RP6530, a dual PI3K δ/γ inhibitor, resulted in a reversal of ruxolitinib resistance. Complementary activity was also observed in HEL-RS cells indicating that a combination of ruxolitinib and RP6530 could have a positive bearing on the clinical outcome in MF patients.

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DR ANTHONY MELVIN CRASTO, Born in Mumbai in 1964 and graduated from Mumbai University, Completed his Ph.D from ICT, 1991,Matunga, Mumbai, India, in Organic Chemistry, The thesis topic was Synthesis of Novel Pyrethroid Analogues, Currently he is working with GLENMARK PHARMACEUTICALS LTD, Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 29 plus yrs, Prior to joining Glenmark, he has worked with major multinationals like Hoechst Marion Roussel, now Sanofi, Searle India Ltd, now RPG lifesciences, etc. He has worked with notable scientists like Dr K Nagarajan, Dr Ralph Stapel, Prof S Seshadri etc, He did custom synthesis for major multinationals in his career like BASF, Novartis, Sanofi, etc., He has worked in Discovery, Natural products, Bulk drugs, Generics, Intermediates, Fine chemicals, Neutraceuticals, GMP, Scaleups, etc, he is now helping millions, has 9 million plus hits on Google on all Organic chemistry websites. His friends call him worlddrugtracker. His New Drug Approvals, Green Chemistry International, All about drugs, Eurekamoments, Organic spectroscopy international,
etc in organic chemistry are some most read blogs He has hands on experience in initiation and developing novel routes for drug molecules
and implementation them on commercial scale over a 29 year tenure till date Aug 2016, Around 30 plus products in his career. He has good knowledge of IPM, GMP, Regulatory aspects, he has several International patents published worldwide . He has good proficiency in Technology transfer, Spectroscopy, Stereochemistry, Synthesis, Polymorphism etc., He suffered a paralytic stroke/ Acute Transverse mylitis in Dec 2007 and is 90 %Paralysed, He is bound to a wheelchair, this seems to have injected feul in him to help chemists all around the world, he is more active than before and is pushing boundaries, He has 9 million plus hits on Google, 2.5 lakh plus connections on all networking sites, 25 Lakh plus views on dozen plus blogs, He makes himself available to all, contact him on +91 9323115463, email amcrasto@gmail.com, Twitter, @amcrasto , He lives and will die for his family, 90% paralysis cannot kill his soul., Notably he has 13 lakh plus views on New Drug Approvals Blog in 212 countries......https://newdrugapprovals.wordpress.com/ , He appreciates the help he gets from one and all, Friends, Family, Glenmark, Readers, Wellwishers, Doctors, Drug authorities, His Contacts, Physiotherapist, etc

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